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US7588909B2 - Method for detecting Streptococcus agalactiae using α-glucosidase activity - Google Patents

Method for detecting Streptococcus agalactiae using α-glucosidase activity Download PDF

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US7588909B2
US7588909B2 US11/660,867 US66086705A US7588909B2 US 7588909 B2 US7588909 B2 US 7588909B2 US 66086705 A US66086705 A US 66086705A US 7588909 B2 US7588909 B2 US 7588909B2
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substrate
medium
reaction medium
enzymatic
streptococcus agalactiae
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US20070292908A1 (en
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Denis Robichon
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Biomerieux SA
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • C12Q1/14—Streptococcus; Staphylococcus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/315—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Streptococcus (G), e.g. Enterococci

Definitions

  • the present invention relates to the field of the detection and identification of Streptococcus agalactiae. More particularly, the invention relates to the use of ⁇ -glucosidase enzymatic substrates for detecting and identifying Streptococcus agalactiae.
  • Streptococcus genus contains numerous species of a very wide spread in streptococci nature, on the skin of the mucous membranes of humans and animals, and are responsible for multiple infections. They are ubiquitous bacteria that are found in the free state in the outside environment (soil, air, water), in the saprophyte state or in the commensal state in humans and animals. They are located in the rhinopharynx for group A, C, G and H streptococci and salivarius, the intestine for group D fetal streptococci and the vaginal cavity for group B streptococci. Their pathogenic role is extremely varied and depends on the species in question and on their location in the organism.
  • Streptococci are Gram+cocci, 0.5 to 1 ⁇ m in diameter, they exhibit grouping in the form of a small chain and are immobile. They are catalase-negative, have a fermentative metabolism, and they are optionally anaerobic and are sensitive to variations in temperature (optimal growth 37° C.) and to variations in pH (optimal pH 7).
  • Streptococcus agalactiae (or streptococcus B) is recognized as one of the main infectious agents responsible for mastitis in cattle. In humans, it is essentially a saprophyte of the female genital tract (vagina), but it is also found in the rhinopharynx and in the intestine, in particular the rectum. In adults, colonization often remains asymptomatic, but Streptococcus agalactiae can be responsible for septicemia, pneumonia, meningitis, arthritis, urinary infections and deep suppurations. In women who are pregnant, or after having given birth, the infection may lead to endometritis and to sterility.
  • the selective media most commonly used are Todd-Hewitt broth, an enrichment broth for searching for group B streptococci in pregnant women.
  • This broth contains various antibiotics that inhibit most gram-negative microorganisms of the accompanying flora, such as nalixidic acid and gentamycin, or nalixidic acid, polymyxin and crystal violet.
  • the antibiotic-supplemented Todd-Hewitt broth must be subcultured on media for searching for streptococci (see CDC (Center for Disease Control) recommendations, MMWR (Morbidity and Mortality Weekly Report), Aug. 16, 2002, Vol. 51, No. RR-11).
  • Lim medium is a variant of Todd-Hewitt broth and it contains 1% of yeast extract, nalixidic acid and colistin.
  • a Columbia agar containing 5% of blood is also used and makes it possible in particular, to demonstrate the ⁇ -hemolytic characteristic of Streptococcus agalactiae.
  • this characteristic is not always apparent: the hemolytic halo around the colonies may be narrow, giving rather the ⁇ -hemolytic, or even ⁇ -hemolytic, appearance.
  • this characteristic becomes clear if, in the area of the Streptococcus agalactiae colonies, there are Staphylococcus aureus colonies (Camp-factor).
  • enzymatic substrates in particular ⁇ -glucosidase enzymatic substrates, for specifically detecting and identifying Streptococcus agalactiae.
  • the enzymatic substrates used by the Streptococcus agalactiae allowing them to be revealed, for example by producing a modification of the coloration of the colonies in the medium when a chromogenic enzymatic substrate is used, with no diffusion of the coloration in the reaction medium and said coloration therefore remaining concentrated at the colonies, but these molecules have no harmful effect on the growth of these bacteria.
  • a subject of the present invention is a method for specifically detecting and identifying Streptococcus agalactiae, characterized in that a reaction medium comprising at least one ⁇ -glucosidase enzymatic substrate is used.
  • the ⁇ -glucosidase enzymatic substrates suitable for the purposes of the invention are any substrate known to those skilled in the art that makes it possible to demonstrate such an enzymatic activity.
  • Such substrates may, for example, be chromogenic or fluorescent and are described, for example, in the article by P. Rice et al. (2000). A rapid biochemical test to aid identification of Mycoplasma mycoides subsp. mycoides small colony (SC) strains. Lett. Appl. Microbiol. 1:70-74, or in the BIOSYNTH catalogue, Substrates and Reagents or in the GLYCOSYNTH catalogue, enzyme substrates catalouge.
  • indoxyl-derivative-based substrates examples include indoxyl-derivative-based substrates, umbelliferone-derivative-based substrates and naphthol-derivative-based substrates.
  • the enzymatic substrate suitable for the purposes of the invention is an indoxyl-derivative-based substrate.
  • indoxyl derivatives examples include 3-indolyl- ⁇ -D-glucopyranoside derivatives, preferably halogenated derivatives of these compounds.
  • halogenated 3-indolyl- ⁇ -D-glucopyranoside derivatives mention may be made of 6-bromo-3-indolyl- ⁇ -D-glucopyranoside, 6-chloro-3-indolyl- ⁇ -D-glucopyranoside, 5-bromo-6-chloro-3-indolyl- ⁇ -D-glucopyranoside, 5-bromo-4-chloro-3-indolyl- ⁇ -D-glucopyranoside and 5-bromo-4-chloro-3-indolyl-N-methyl- ⁇ -D-glucopyranoside, the latter compound being particularly preferred.
  • the reaction medium as used in the method of the invention is therefore a detection reaction medium due to the presence of the enzymatic substrate.
  • This reaction medium can be used either as a visualization medium only, or as a culture and visualization medium.
  • the culturing of the microorganisms is carried out before inoculation and, in the second case, the reaction medium also constitutes the culture medium.
  • the reaction medium may be solid, semi-solid or liquid.
  • solid or semi-solid medium is intended to mean, for example, a gelled medium.
  • Agar is the conventional solid medium in microbiology for culturing microorganisms, but it is possible to use gelatin or agarose.
  • gelatin or agarose A certain number of preparations are commercially available, for instance Columbia agar, Trypcase-soy agar, MacConkey agar, Sabouraud agar or, more generally, those described in the Handbook of Microbiological Media (CRC Press).
  • the amount of agar in the reaction medium is from 2 to 40 g/l.
  • the amount of agar is preferably from 9 to 25 g/l, more preferably from 12 to 14 g/l.
  • the amount of agar is preferably from 2 to 6 g/l.
  • the enzymatic substrates of the invention can be used in a wide pH range, in particular between pH 5.5 and 10.
  • the concentration of the enzymatic substrate in the reaction medium is between 10 and 2000 mg/l, preferably between 50 and 500 mg/l, more preferably between 100 and 400 mg/l, which constitutes a preferred embodiment of the invention.
  • the concentration of the enzymatic substrate in the medium within this range, according to the substrate chosen.
  • the enzymatic substrate used is 5-bromo-4-chloro-3-indolyl-N-methyl- ⁇ -D-glucopyranoside
  • a concentration between 130 and 300 mg/l is preferred.
  • reaction medium that can be used for the purposes of the invention may also comprise other components of use for improving the specificity and the sensitivity of the method of the invention.
  • the reaction medium may also contain a mixture of inhibitors for inhibiting or limiting the growth of unwanted strains, such as false-positive strains, for example Candida or Staphylococcus saprophyticus, without modifying the detection sensitivity of the medium.
  • unwanted strains such as false-positive strains, for example Candida or Staphylococcus saprophyticus
  • the reaction mixture may contain a mixture of antibiotics.
  • antibiotics to the reaction medium allows, inter alia, a gain in time since the identification of Streptococcus agalactiae is carried out directly.
  • antibiotics examples include aztreonam and amphotericin B. These antibiotics are commercially available from ICN, Squibb or Sigma.
  • the reaction medium used in the method for specifically detecting and identifying Streptococcus agalactiae also comprises a mixture of aztreonam and amphotericin B.
  • the amount of each antibiotic in the reaction medium varies according to the antibiotic concerned, and will be readily determined by those skilled in the art.
  • the concentration of aztreonam may be between 0.01 and 0.08 g/l and the concentration of amphotericin B may be between 0.002 and 0.006 g/l.
  • a preferred mixture of these antibiotics contains 0.064 g/l of aztreonam and 0.004 g/l of amphotericin B.
  • the reaction medium used in the method of the invention may also comprise at least one other substrate specific for an enzymatic activity other than that detected by the ⁇ -glucosidase substrate.
  • the enzymatic hydrolysis of the other substrate(s) generates a detectable signal different than the signal detected by means of the ⁇ -glucosidase substrate, such as, for example, different colored or fluorescent products, so as to allow the demonstration, such as the detection and/or the identification and/or the quantification, of Streptococcus agalactiae by improving the specificity of demonstration.
  • ⁇ -cellobiosidase substrates By way of another specific substrate, mention may be made of ⁇ -cellobiosidase substrates, ⁇ -glucosidase substrates, ⁇ -glucosaminidase substrates and any other enzymatic substrate known to those skilled in the art that makes it possible to eliminate false positives.
  • the reaction medium also comprises at least one other substrate for different enzymatic activity, preferably chosen from ⁇ -cellobiosidase substrates and ⁇ -glucosaminidase substrates.
  • ⁇ -cellobiosidase substrate such as 6-chloro-3-indolyl- ⁇ -D-cellobioside makes it possible to eliminate false-positive species, such as Enteroccocus faecalis, Listeria monocytogenes and Enterobacter claocae, without degrading the sensitivity of detection of the ⁇ -glucosidase activity of Streptococcus agalactiae.
  • glucosaminidase substrate such as 6-chloro-3-indolyl- ⁇ -N-acetylglucosaminide or 5-bromo-6-chloro-3-indolyl- ⁇ -N-acetylglucosaminide makes it possible to eliminate the detection of Enteroccocus faecalis, Enteroccocus faecium and Enterobacter claocae.
  • the concentration of the other specific enzymatic substrate is generally between 0.01 and 2 g/l. Those skilled in the art will be able to readily determine such a concentration according to the substrate used.
  • 6-chloro-3-indolyl- ⁇ -D-cellobioside or 6-chloro-3-indolyl- ⁇ -N-acetylglucosaminide When 6-chloro-3-indolyl- ⁇ -D-cellobioside or 6-chloro-3-indolyl- ⁇ -N-acetylglucosaminide is used, its concentration in the medium is preferably 0.4 g/l.
  • the reaction medium may also comprise one or more elements in combination, such as amino acids, peptones, carbohydrates, nucleotides, minerals, vitamins, surfactants, buffers, phosphate salts, ammonium salts, sodium salts or metal salts. Examples of media are described in the applicant's patent applications EP 656 421 and WO99/09 207.
  • the incubation temperature may be 37° C.
  • the incubation atmosphere it is preferably aerobic.
  • the revealing is carried out with the naked eye by visualization of a change in coloration that does not diffuse in the reaction medium and is therefore concentrated at the colonies.
  • the fluorescence-reading devices known to those skilled in the art are used.
  • the biological samples to be analyzed are any clinical sample liable to contain Streptococcus agalactiae, such as a vaginal specimen, a urine specimen or any other sample of which the analysis may aid a clinician in reaching a diagnosis.
  • the reaction media were prepared by mixing heart-brain extract (4.84 g/l; Solabia), meat infusion (1.96 g/l; Solabia), biothione (1 g/l; Solabia), biotrypcase (7.2 g/l; Solabia), sodium carbonate (0.3 g/l; VWR), sodium pyruvate (2 g/l; Fluka), HEPES buffer (0.4 g/l; Sigma), lactalbumin peptone (2 g/l; DMV), glucose (1 g/l; Merck), American agar (2 g/l; Sobigel) and European agar (12 g/l; Roko).
  • an ⁇ -glucosidase enzymatic substrate as indicated below is added at a rate of 0.1 g/l, followed by cooling in a water bath at 50° C.
  • the media were then poured into a Petri dish for subsequent inoculation with bacterial strains.
  • Table 2 gives the number of strains detected (considered to be positive when I ⁇ 0.6) as a function of the concentration of substrate.
  • control medium 1 control medium corresponding to the medium of example 1 with GreenA- ⁇ -Glu, modified as indicated above,
  • medium 2 400 mg/l of 6-chloro-3-indolyl- ⁇ -D-cellobioside (Rose- ⁇ -cellobioside),
  • medium 3 400 mg/l of 6-chloro-3-indolyl- ⁇ -N-acetylglucosaminide (Rose- ⁇ -NAGlu) and 0.5 g/l of N-acetylglucosaminide.
  • strains are all derived from the applicant's collection.
  • a medium according to the invention prepared as described in the above examples, but containing 0.130 g/l of GreenA- ⁇ -Glu, 0.250 g/l of Rose- ⁇ -D-cellobioside, 0.064 g/l of aztreonam and 0.004 g/l of amphotericin B ( ⁇ -Glu medium), was used.
  • Granada medium As medium for comparison, the Granada medium (ref 10 077, BIOLYS, France) (Granada medium) was used.
  • results are expressed as % of correct diagnosis relative to all the tests in terms of sensitivity and specificity, and are given in table 4 below, the % sensitivity corresponding to the number of true positives detected on the medium divided by the total number of true positives to be detected (*100) and the % specificity corresponding to the number of true negatives detected on the medium divided by the total number of true negatives to be detected (*100).
  • results indicated in this table demonstrate the improvement in the sensitivity of detection of Streptococci B using the method of the invention. Moreover, they also show that the detection medium of the invention also has good specificity, which specificity is improved after enrichment due to a passage in Todd-Hewitt broth for 18-24 hours at 35-37° C. with or without 5% CO 2 before inoculation of the agar (see CDC (Center for Disease Control) recommendations, MMWR (Morbidity and Mortality Weekly Report), Aug. 16, 2002, vol. 51, No. RR-11).
  • Each swab was emulsified in 1 ml of sterile physiological saline and 100 ⁇ l of this solution were deposited, firstly, onto a Columbia agar containing 5% of horse blood and, secondly, onto the medium using the method of the invention. Moreover, 100 ⁇ l of the above solution were used to inoculate a Todd-Hewitt broth. After incubation for 20 hours at 37° C. and under aerobic conditions, the blood-agar and the medium of the invention were inoculated using the Todd-Hewitt broth, and then incubated at 37° C. for 20 h under aerobic conditions.
  • 112 were inoculated onto the agar media, firstly directly from the suspension in physiological saline and, secondly, after enrichment in Todd-Hewitt broth.
  • the remaining 22 samples were inoculated onto the agar media only directly from the suspension in physiological saline.
  • results in the table show that the medium, used with clinical samples, has good sensitivity (18/20 Streptococcus agalactiae detected) and that the specificity of detection is improved compared with a standard medium (10 false + results against 24 on the Columbia blood medium), these results being substantially equivalent to those obtained using the laboratory strains.

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US11/660,867 2004-09-16 2005-09-14 Method for detecting Streptococcus agalactiae using α-glucosidase activity Active 2026-04-10 US7588909B2 (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
FR0452066A FR2875240B1 (fr) 2004-09-16 2004-09-16 Procede de detection de streptococcus agalactiae en utilisant l'activite alpha-glucosidase
FR0452066 2004-09-16
PCT/FR2005/050740 WO2006032810A1 (fr) 2004-09-16 2005-09-14 PROCEDE DE DETECTION DE STREPTOCOCCUS AGALACTIAE EN UTILISANT L'ACTIVITE α-GLUCOSIDASE

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EP (1) EP1789573B1 (fr)
JP (1) JP4999693B2 (fr)
CN (1) CN101027406B (fr)
AT (1) ATE426039T1 (fr)
DE (1) DE602005013403D1 (fr)
ES (1) ES2323428T3 (fr)
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KR20110017881A (ko) * 2008-05-15 2011-02-22 토피겐 파마슈티컬스 인코포레이티드 감염 및 신생세포 증식 치료용 올리고뉴클레오티드
FR2937052A1 (fr) * 2008-10-08 2010-04-16 Biomerieux Sa Milieu reactionnel pour les bacteries staphylococcus aureus
FR2936816B1 (fr) * 2008-10-08 2013-03-22 Biomerieux Sa Milieu reactionnel pour les bacteries staphylococcus aureus resistantes a la meticilline (mrsa)
US8497086B2 (en) * 2009-08-13 2013-07-30 Biomereux Reaction medium for methicillin-resistant Staphylococcus aureus (MRSA) bacteria
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US20140113321A1 (en) * 2012-03-21 2014-04-24 Empire Technology Development Llc Identification of cellulolytic microorganism contamination in food and other materials
WO2015092481A1 (fr) * 2013-12-16 2015-06-25 Compagnie Gervais Danone Procédé de dénombrement différentiel de bactéries lactiques en mélange dans un produit alimentaire
CN107287275B (zh) * 2016-03-31 2022-02-01 启新生物科技有限公司 培养基、包含其的试剂盒、及其应用
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US8415393B2 (en) * 2007-05-22 2013-04-09 Wisconsin Alumni Research Foundation Anti-bacterial drug targeting of genome maintenance interfaces

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JP4999693B2 (ja) 2012-08-15
ES2323428T3 (es) 2009-07-15
DE602005013403D1 (de) 2009-04-30
EP1789573B1 (fr) 2009-03-18
WO2006032810A1 (fr) 2006-03-30
ATE426039T1 (de) 2009-04-15
JP2008513001A (ja) 2008-05-01
EP1789573A1 (fr) 2007-05-30
US20070292908A1 (en) 2007-12-20
FR2875240B1 (fr) 2006-11-17

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